Using both biochemical and molecular genetic approaches, this investigator's laboratory is involved in studying eukaryotic translation initiation particularly with regard to the interaction of translation initiation factors with other components of the translation initiation machinery during formation of the 40S and 80S initiation complexes. In this grant proposal, the following areas will be investigated. (a) Following scanning of the mRNA and recognition of the AUG codon by the 40S preinitiation complex to form the 40S initiation complex (40S.elF3.mRNA*Met-tRNA.elF2.GTP), eIF5 interacts with the 40S initiation complex to promote the hydrolysis of bound GTP. Genetic evidence in the yeast S. cerevisiae indicates that elF5-promoted GTP hydrolysis is tightly coupled to the selection of the AUG codon by the 40S preinitiation complex. This process will be studied in vitro using the purified mammalian initiation factors. The role of interaction of elF5 with other initiation factors involved in the formation of the 40S preinitiation and initiation complexes in coupling GTP hydrolysis to the AUG selection process will be investigated. (b) Mammalian initiation factor elF3 consists of five "core" subunits which are conserved in all known eukaryotes, and five "non-core" subunits which do not have structural homologues in the budding yeast. The function of the core and non-core subunits will be investigated by co-expressing the elF3 subunits in various combinations in the baculoviral expression system. The resultant complexes will be purified and tested for the known elF3 reactions in vitro. The in vivo function of the non-core subunits will be analyzed using the molecular genetic techniques of the fission yeast, S. pombe, an organism which encodes four of the five non-core subunits. (c) Translation initiation factor elF6, a 26 kDa protein, binds to 60S ribosomal subunits and prevents the association between the 40S and 60S ribosomal subunits. In yeast cell free extracts, elF6 is boUnd to the free 60S ribosomal subunits in the cytoplasm. However, the 80S ribosomes or the polysomes do not contain any bound elF6 indicating that elF6 must be released from the 60S particles prior to 80S complex formation. elF6 is also phosphorylated by an elF6-specific kinase in yeast and mammalian cells. The role of phosphorylation of elF6 andfor interaction of eIF6 with other known or novel proteins in the release of elF6 from the 60S.elF6 complex will be investigated.